CN107630061A - The preparation method of Yak Bone NTx albumen - Google Patents
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Abstract
The preparation method of Yak Bone NTx albumen provided by the invention, Yak Bone is carried by pretreatment, sour lyase, centrifuged, the technique such as freezes the higher Yak Bone NTx protein product of purity is prepared.Due to having been carried out preferably to decalcification degreasing condition, extraction enzyme concentration by experiment in the present invention, and structure verification is carried out to Yak Bone NTx albumen using means such as UV, FT IR, SEM, it specify that prepared Yak Bone NTx protein specificity structural integrity, purity is high, is capable of the comprehensive utilization value of significant increase edibility bone.
Description
Technical field
The invention belongs to bone processing technique field, more particularly to a kind of preparation method of Yak Bone NTx albumen.
Background technology
The edibility bone aboundresources in China, at present China's live stock and fowl bone annual production are up to more than 1,200 ten thousand tons, account for world wide production
1/3, convert into about 2,000,000 tons of animal proteins, the year albumen demand of 75,000,000 people can be met.China steps into aging society at once
Meeting, osteoporosis turn into the elderly in face of maximum chronic threat illness.It is limited in population pressure and breeding environment bearing capacity
Under conditions of, strengthen the comprehensive development and utilization to bone resource, there is important economy, society and environmental benefit.
Yak (Bos grunniens) is rare ox kind in the world, and China is to possess the most country of yak in the world,
About 14,000,000, quantity accounts for the total amount of livestock on hand 16.67% of beef cattle.Yak butchering amount about 3,000,000 in 2015, produce Yak Bone about
100000 tons.And because yak is that seasonality is butchered, the short time can produce substantial amounts of ox bone, most of in the form of primary product
Sell or directly discarded, altitude environment is exerted heavy pressures on.Containing abundant nutrient in Yak Bone, have to health
Good nutrition enhancing and therapeutic effect.Deepen the research to Yak Bone, for improving household's actions, protection highlands ring
Border, it is significant.
The product developed currently with Yak Bone is only limited to carry out simply based on traditional Chinese medicine, Tibetan medicine theory
The extensive product of processing, such as yak bone powder, Production of Marrow of Yak Sauce, yak bone carving etc., profound utilization and high added value processing
Seldom.Whole mineral compositions containing almost needed by human body in ox bone, including the necessary trace element of human body, and wherein institute
Containing rich in nutrition content such as albumen, grease, polysaccharide.Therefore, it is comprehensive to be directed to contained mineral matter, protein, fat in ox bone
It is analyzed and evaluated Deng nutrition composition, for comprehensively utilizing ox bone resource, develops new high value added product, there is important meaning
Justice.Yak butchering amount about 3,000,000 in 2015, yak carcass weight average out to 123.0kg/ heads, trunk yield is about 400,000 tons, ox
Meat production is close to 300,000 tons, about 100,000 tons of ox bone.In recent years, the comprehensive utilization for the meat of yak, breast, skin, bone there has also been not
Few research.Containing abundant mineral matter element in Yak Bone, the person in middle and old age's sclerotin that grows of Children and teenager bone is dredged
Loose disease prevents have important nutrition and therapeutic potential, and DEVELOPMENT PROSPECT is wide.
The content of the invention
It is an object of the invention to solve at least the above and/or defect, and provide at least will be described later it is excellent
Point.
The present invention provides a kind of preparation method of Yak Bone NTx albumen, and it comprises the following steps：
Step 1：Will freezing Yak Bone raw material by preferably with after cleaning pass through hard bone crusher in crushing be made Yak Bone
Broken material；
Step 2：The Yak Bone broken material is subjected to cleaning impurity elimination；
Step 3：After through the Yak Bone broken material of over cleaning impurity elimination using ether defatting processing, EDTA is reused
Or HCl method carries out decalcification and handles to obtain degreasing decalcification Yak Bone；
Step 4：Degreasing decalcification Yak Bone is dissolved using acetic acid and adds pepsin and carries out enzymolysis processing；
Step 5：Gained supernatant after enzymolysis is added into NaCl solution to be saltoutd；
Step 6：Product is centrifuged to obtain sediment after saltouing；
Step 7：Sediment is freezed and obtains Yak Bone NTx albumen finished product.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 1,
The cryogenic temperature of the freezing Yak Bone raw material is -18 DEG C；
The particle diameter of the Yak Bone broken material is 10-20mm.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 3,
The ether defatting processing is to carry out degreasing using 4~5h of ether low temperature reflux；
The decalcification processing is to use the HCl of 0.25~0.50mol/L EDTA or concentration for 0.5~1.0mol/L.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 4,
The weight ratio of the acetic acid solution and the degreasing decalcification Yak Bone is 1：8-10；
The molar concentration of the acetic acid solution is 0.3-0.5mol/L；
The weight ratio of the pepsin and the acetic acid solution is 1-1.8：100；
The enzymolysis processing is to carry out shaking extraction, time 24h-120h.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 5, the NaCl solution
Molar concentration be 0.5-1.0mol/L.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 6, centrifuging temperature 4
DEG C, centrifugal rotational speed 10000rmp, centrifugation time 30min.
Preferably, the preparation method of described Yak Bone NTx albumen, in the step 7, freeze temperature is -40
DEG C -- 47 DEG C, freeze-drying time 12h-24h, vacuum is -0.08Mpa--0.09Mpa.
Yak Bone is carried by pretreatment, sour lyase by above step, centrifuged, freeze etc. technique be prepared it is pure
The higher Yak Bone NTx protein product of degree.Due to being entered in this patent by experiment to decalcification degreasing condition, extraction enzyme concentration
Go preferably, and structure verification has been carried out to Yak Bone NTx albumen using means such as UV, FT-IR, SEM, specify that made
Standby Yak Bone NTx protein specificity structural integrity, purity is high, is capable of the comprehensive utilization value of significant increase edibility bone.
The present invention comprises at least following beneficial effect：
The method that the present invention is carried using sour lyase, obtain in prepared Yak Bone NTx albumen Gly ratios most
It is high.
The ultraviolet absorption peak 234nm of Yak Bone NTx albumen is consistent with ox bone collagen, meets ox bone collagen egg
White Absorption Characteristics.
Prepared Yak Bone NTx albumen all possesses preferable secondary structure, in amide Ⅰ and the a- of the zone of acid amides II
Spiral ratio is high, and the collagen structure integrality extracted is more preferable.Detected by SEM and SDS-PAGE, Yak Bone NTx
Albumen is consistent with the feature that the ox bone collagen albumen that other researchers extract is presented.
Collagen prepared by Yak Bone meets the architectural feature of I type ox bone collagen albumen, simply in some amino acid
It is different among content and DSC results, but otherness is not notable.
Preparation method of the invention is simple, production efficiency is high, economic benefit protrudes, and produces Yak Bone collagen and can be used for giving birth to
Tetramune, organizational project, functional food etc..
Due to having carried out structure verification to collagen optimal extraction technology, and to its structure.
Present invention process is easy to operate, and reliable guide is provided for large-scale production.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of Yak Bone NTx albumen provided by the invention；
Fig. 2 is the different degreasings in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
Method effect contrast figure；
Fig. 3 is that the different salt in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention are dense
Spend the influence comparison diagram to collagen yield；
Fig. 4 is the different stomach eggs in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
White influence comparison diagram of the enzyme concentration to collagen yield；
Fig. 5 is the type glue of Yak Bone I in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
The ultraviolet spectrogram of former albumen；
Fig. 6 is the type glue of Yak Bone I in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
The FTIR spectrum figure of former albumen；
Fig. 7 is the type glue of Yak Bone I in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
The Heat Flow Analysis figure of former albumen；
Fig. 8 is the type glue of Yak Bone I in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
The electrophoresis result figure of former albumen；
Fig. 9 is the type glue of Yak Bone I in one embodiment of the preparation method of Yak Bone NTx albumen provided by the invention
The micro-structure diagram of former albumen.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of individual other elements or its combination.
As shown in figure 1, the preparation method of Yak Bone NTx albumen provided by the invention includes：
Step 1：Yak Bone raw material is freezed by preferably with after cleaning, passing through hard bone crusher in crushing；
Step 2：The purpose of pretreatment is mainly cleaned and removes caused blood, disintegrating slag, attachment effect after crushing；
Step 3：After broken bone after crushing is first passed through into ether defatting, reuse EDTA or HCl and broken bone is carried out at decalcification
Reason；
Step 4：Product after degreasing decalcification is dissolved using acetic acid and adds pepsin enzymolysis；
Step 5：Gained supernatant after enzymolysis is added into finite concentration NaCl solution to be saltoutd；
Step 6：Product is centrifuged to obtain sediment after saltouing；
Step 7：Sediment is freezed and obtains Yak Bone NTx albumen finished product.
Precrushing processing described in above-mentioned steps one includes：Used raw material is -18 DEG C of freezing state, and Yak Bone needs
To through by hard bone crusher in crushing and crushing, grinding particle size is controlled in 10~20mm；
Pre-treatment step described in above-mentioned steps two includes：Rinse, clear up impurity elimination, remove attachment；
Degreasing, decalcification step described in above-mentioned steps three include：Broken bone after cleaning uses 4~5h of ether low temperature reflux
Carry out degreasing；0.25~0.50mol/L EDTA or concentration is used to carry out decalcification processing for 0.5~1.0mol/L HCl；
Dissolving, extraction step described in above-mentioned steps four include：The broken bone after degreasing decalcification, add 0.3~0.5mol/
L acetic acid solution, the pepsin for adding 1~1.8% carry out enzymolysis and extraction, solid-liquid ratio 1:8~10 shake extraction 24h~
5d；
Salting-out step described in above-mentioned steps five includes：The NaCl solution for adding 0.5~1.0mol/L into supernatant is entered
Row is saltoutd；
Centrifugation step described in above-mentioned steps six includes：At 4 DEG C, centrifugal rotational speed is that 10000rmp centrifuges 30min centrifugations
Precipitated afterwards；
Step of freeze drying described in above-mentioned steps seven includes：Under the conditions of vacuum -0.08~-0.09MPa, -40~-47 DEG C
When lyophilized 12~24h obtain Yak Bone NTx albumen.
Embodiment one
1st, -18 DEG C of 10 kilograms of frozen bone raw materials are chosen, after its impurity is rinsed well with clear water first, are broken by hard bone
Broken crusher machine, which will pass through, to be crushed, and grinding particle size is controlled in 10mm；
2nd, the bone after crushing rinsed using clear water, clear up impurity elimination, remove attachment；
3rd, broken bone after cleaning carries out degreasing using ether low temperature reflux 4h；Use 0.25mol/L EDTA or concentration for
0.5mol/L HCl carries out decalcification processing；
4th, the broken bone after degreasing decalcification, 0.5mol/L acetic acid solution is added, the pepsin for adding 1% carries out enzyme
Solution extraction, solid-liquid ratio 1:8 shake extraction 24h；
5th, the salting-out step described in above-mentioned steps five includes：The NaCl solution that 0.5mol/L is added into supernatant carries out salt
Analysis；
6th, the centrifugation step described in above-mentioned steps six includes：At 4 DEG C, centrifugal rotational speed be 10000rmp centrifuge 30min from
Precipitated after the heart；
7th, the step of freeze drying described in above-mentioned steps seven includes：Under the conditions of vacuum -0.08MPa, lyophilized 12h at -40 DEG C
Obtain Yak Bone NTx albumen.
Embodiment two
1st, using the freezing state that raw material is -18 DEG C, Yak Bone, which needs to pass through by hard bone crusher in crushing, to be crushed,
Grinding particle size is controlled in 20mm；
2nd, the bone after crushing rinsed using clear water, clear up impurity elimination, remove attachment；
3rd, broken bone after cleaning carries out degreasing using ether low temperature reflux 5h；Use 0.50mol/L EDTA or concentration for
1.0mol/L HCl carries out decalcification processing；
4th, the broken bone after degreasing decalcification, 0.5mol/L acetic acid solution is added, adds 1.8% pepsin progress
Enzymolysis and extraction, solid-liquid ratio 1:10 shake extraction 5d；
5th, the NaCl solution that 1.0mol/L is added into supernatant is saltoutd；
6th, at 4 DEG C, centrifugal rotational speed is to be precipitated after 10000rmp centrifuges 30min centrifugations；
7th, under the conditions of vacuum -0.09MPa, lyophilized 24h obtains Yak Bone NTx albumen at -47 DEG C.
Detect verification experimental verification：
1st, degreasing condition selects
As shown in Figure 2, under the conditions of different disposal, ether low temperature reflux shows optimal degreasing effect, is ground with other
The person of studying carefully tests that result is similar, selects degreasing agent of this condition as Yak Bone NTx protein extraction.
2nd, decalcification condition selects
Influence of the table 1-1 differences decalcifying agent to collagen yield
Inorganic matter contained by aggregate after degreasing is up to more than 68%, it is necessary to could obtain purer collagen egg after removing
In vain, because the main component of inorganic mineral is calcium phosphate and calcium carbonate, usually using hydrochloric acid or phosphoric acid as decalcifying agent in production.
From table 1-1, EDTA decalcification effect is superior to HCl under the conditions of comparable sodium, due to HCl and bone calcium reaction speed by
HCl concentration has a great influence, and EDTA can form stable chelate as chelate divalent metal agent with bone calcium.0.50 mol/L
When EDTA is as decalcifying agent, the yield highest of collagen, 0.25mol/L EDTA yield is slightly below 0.50 mol/L EDTA
Decalcifying agents of group, this experiment selection 0.25mol/L EDTA or selection the 1.0mol/L HCl as ox bone collagen protein extraction.
3rd, salting point selects
Using 2.6mol/L NaCl concentration, ox bone NTx albumen is obtained by being freeze-dried.From the figure 3, it may be seen that with
The increase of NaCl concentration, collagen yield is presented first to increase and reduced afterwards, in NaCl concentration>There is increase again after 0.9mol/L to become
Gesture；When NaCl concentration is 0.9mol/L, collagen yield is significantly higher than other each groups (P<0.05).Therefore this experiment selection
Salting points of the 0.9mol/L NaCl as Yak Bone NTx protein extraction.
4th, the optimization of enzyme addition
During using Collagen Extraction with Enzyme albumen, extraction conditions must be strictly controlled, such as the species of enzyme, enzymolysis time and temperature
Deng.During general extraction animal protein, pepsin is more conventional, and its mild condition is easily controllable.There is scholar to 4 kinds of conventional eggs
White enzyme also indicates that affinity of the pepsin with trypsase to ox bone albumen to the enzymolysis kinetics result of study of ox bone albumen
Larger, the Vmax of alkali protease and pepsin is larger.Therefore, the present invention is also chosen by pepsin assisted extraction ox bone collagen
Albumen.As shown in Figure 4, when pepsin addition is 1.6%, the rate of collagen is significantly higher than other each groups (p<
0.05).Therefore this experiment selection pepsin addition is 1.6%.
5th, amino acid composition and content
From table 1-2, Yak Bone NTx albumen has the compositing characteristic of typical NTx albumen.Wherein, TAA
Ratio shared by respectively 652.4mg/g, EAA is 20.1%；Gly contents are most, account for the 25.9% of TAA, be secondly Pro,
Glu、Ala、Arg、Asp；Collagen amino acids characteristic (Gly, Lys, Pro) total amount reaches 273mg/g；Do not contain Trp,
Cys, Met and Tyr are 3 kinds of amino acid that content is minimum in Yak Bone NTx albumen.
Table 1-2 Yak Bone NTx histone amino acid contents (mg/g)
Table 6-3 Amino acid concentration of collagen from Gannan yak bone
(mg/g)
6th, UV Spectroscopic analysis results
Due to not containing Trp in Yak Bone NTx albumen, Tyr contents are relatively low, so collagen solution exists
Below 240nm has and does not have absworption peak substantially in more than 240nm compared with strong absworption peak.
As shown in figure 5, Yak Bone NTx albumen reaches absorption maximum at 234nm, the glue reported with pertinent literature
Former albumen at 230nm or so places there is obtained the maximum absorption to match.But exist with the ox bone NTx albumen among studying
Maximum absorption band at 228nm still slightly has gap, and reason is due to that the difference of extracting mode causes amino acid the Nomenclature Composition and Structure of Complexes
Cause.
7th, FT-IR analysis results
Numerous studies report utilizes FT-IR analysing protein secondary structures, (band of acid amides I, II, III), egg in IR bands of a spectrum
Bai Junhui produces characteristic absorption, amide Ⅰ (1700~1600cm-1) due to its ripe spectral peak point out technology turn into it is most strong
Study the bands of a spectrum region of protein structure.
It will be appreciated from fig. 6 that Yak Bone NTx albumen is in 3400cm-1It is nearby acid amides A bands, is the characteristic absorption of protein
Peak；Amide Ⅰ is both present in 1651.01cm-1Place, is to be caused by the C-O stretching vibrations in collagen polypeptide skeleton, with collagen
The secondary structure of albumen is relevant；1450~1600cm-1It is the characteristic absorption frequency of the band of acid amides II, Yak Bone NTx albumen is only
In 1579.65cm-1There is maximum absorption band；Gly, Pro, Hyp content in collagen is of a relatively high, therefore the type of Yak Bone I
Collagen is in 1200~1400cm-1Place has the unexistent absworption peak of other protein, is bent by C-N stretching vibrations and N-H
Vibration causes, and is the CH for belonging to Gly skeletons and Pro side chains2The embodiment at rocking vibration peak；Collagen infrared spectrum feature
Synthesis shows to remain a large amount of triple-helix structures in Yak Bone NTx albumen.
8th, dsc analysis
The Td of Yak Bone NTx albumen is 40.12 DEG C and 40.94 DEG C (Fig. 7) respectively, high compared to pigskin collagen 4 DEG C
Left and right, and extract ox bone collagen protein data with other scholars and differ 3 DEG C, it is close with the collagen data of ox-hide.This may
Related to the content of imino group in collagen, the Td of collagen is related to the content of its hydroxyproline and proline, the two
Content is higher, and the stability of collagen is higher.
9th, SDS-PAGE results
Polyacrylamide gel electrophoresis (SDS-PAGE) analysis is carried out to the collagen extracted in Yak Bone.
As shown in Figure 8.Yak Bone NTx albumen is the tripolymer of NTx protein alpha chain at higher than 250kDa, i.e.,
γ chains, about there is a β band at 200kDa；About there is α at 110kDa1、α2Two electrophoretic bands；Yak Bone NTx albumen
(YBC) α chain bands are relatively shallow thin and small molecular protein band without below 70kDa, show Yak Bone NTx albumen
Protein structure is relatively more complete, without small pointHydrolytic collagen and polypeptide.Therefore, infer that extracted collagen is protected substantially
Complete triple helix structure is held.
10th, microstructure
It is as shown in Figure 9 to Yak Bone NTx albumen microstructure observing using ESEM.The top view of collagen
Show relatively uniform regular, smooth porous network structure with profile, the size in its aperture with preparation process
Water content is relevant；Containing fibrous filament, after 1000 times of amplification, filamentary structure is obvious, and this is collagen molecules
The formed collagenous fibres of aggregation, its formation are relevant with the triple-helix structure of collagen molecules.Yak Bone NTx albumen knot
Structure is evenly distributed, and illustrates to maintain the original fiber knot of collagen substantially in extraction process.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details and shown here as the legend with description.
Claims (7)
1. the preparation method of Yak Bone NTx albumen, it is characterised in that comprise the following steps：
Step 1：By freezing Yak Bone raw material by preferably being crushed with after cleaning by the obtained Yak Bone of hard bone crusher in crushing
Material；
Step 2：The Yak Bone broken material is subjected to cleaning impurity elimination；
Step 3：After through the Yak Bone broken material of over cleaning impurity elimination using ether defatting processing, EDTA or HCl is reused
Method carry out decalcification handle to obtain degreasing decalcification Yak Bone；
Step 4：Degreasing decalcification Yak Bone is dissolved using acetic acid and adds pepsin and carries out enzymolysis processing；
Step 5：Gained supernatant after enzymolysis is added into NaCl solution to be saltoutd；
Step 6：Product is centrifuged to obtain sediment after saltouing；
Step 7：Sediment is freezed and obtains Yak Bone NTx albumen finished product.
2. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 1,
The cryogenic temperature of the freezing Yak Bone raw material is -18 DEG C；
The particle diameter of the Yak Bone broken material is 10-20mm.
3. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 3,
The ether defatting processing is to carry out degreasing using 4~5h of ether low temperature reflux；
The decalcification processing is to use the HCl of 0.25~0.50mol/L EDTA or concentration for 0.5~1.0mol/L.
4. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 4,
The weight ratio of the acetic acid solution and the degreasing decalcification Yak Bone is 1：8-10；
The molar concentration of the acetic acid solution is 0.3-0.5mol/L；
The weight ratio of the pepsin and the acetic acid solution is 1-1.8：100；
The enzymolysis processing is to carry out shaking extraction, time 24h-120h.
5. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 5, institute
The molar concentration for stating NaCl solution is 0.5-1.0mol/L.
6. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 6, from
Heart temperature is 4 DEG C, centrifugal rotational speed 10000rmp, centrifugation time 30min.
7. the preparation method of Yak Bone NTx albumen as claimed in claim 1, it is characterised in that in the step 7, freeze
Dry temperature is -40 DEG C -- 47 DEG C, freeze-drying time 12h-24h, vacuum is -0.08Mpa--0.09Mpa.
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| CN109349419A (en) * | 2018-10-09 | 2019-02-19 | 王书敏 | A kind of compound Yak Bone collagen protein peptide powder for repairing human body cell |
| CN109349419B (en) * | 2018-10-09 | 2022-04-01 | 王书敏 | Compound yak bone collagen peptide powder for repairing human body cells |
| CN111254177A (en) * | 2020-02-17 | 2020-06-09 | 遵义医学院附属医院 | Method for extracting protein from bone marrow |
| CN112410392A (en) * | 2020-11-11 | 2021-02-26 | 武汉盛世伟度生物科技有限公司 | Extraction method and application of type I collagen |
| CN112501229A (en) * | 2020-12-14 | 2021-03-16 | 广州天启生物科技有限公司 | Production process of bovine bone collagen peptide |
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